Bianca de Oliveira, Wanessa M Goes, Frederico C Nascimento, Juliana B T Carnielli, Eden R Ferreira, Alex Fiorini de Carvalho, Pablo Victor Mendes Dos Reis, Milton Pereira, Tiago Queiroga Nery Ricotta, Liliane Martins Dos Santos, Renan Pedra de Souza, Diego Esteban Cargnelutti, Jeremy C Mottram, Santuza R Teixeira, Ana Paula Fernandes, Ricardo T Gazzinelli
{"title":"一种含有重复结构域的新型利什曼原虫抗原的特性及其作为预防性和治疗性疫苗的潜在用途。","authors":"Bianca de Oliveira, Wanessa M Goes, Frederico C Nascimento, Juliana B T Carnielli, Eden R Ferreira, Alex Fiorini de Carvalho, Pablo Victor Mendes Dos Reis, Milton Pereira, Tiago Queiroga Nery Ricotta, Liliane Martins Dos Santos, Renan Pedra de Souza, Diego Esteban Cargnelutti, Jeremy C Mottram, Santuza R Teixeira, Ana Paula Fernandes, Ricardo T Gazzinelli","doi":"10.1128/msphere.00097-25","DOIUrl":null,"url":null,"abstract":"<p><p>Human visceral leishmaniasis (HVL) is the second most lethal tropical parasitic disease. Currently, no prophylactic or therapeutic vaccines exist for HVL. Thus, the development of an efficacious vaccine is still needed. We previously performed an immunoproteomics analysis on <i>Leishmania amazonensis</i> parasite extracts to identify immunodominant antigens recognized by the sera of vaccinated and protected mice. Among the identified antigens, we discovered a novel, previously unstudied repetitive protein, initially annotated in <i>Leishmania</i> genomes as a kinetoplast-associated protein-like protein from <i>Leishmania infantum</i> (LinKAP), containing conserved domains (trichohyalin-plectin-homology [TPH] and TolA) that are associated with other mitochondrial proteins. LinKAP sequences are conserved across trypanosomatids, including <i>Endotrypanum, Leishmania,</i> and <i>Trypanosoma</i> species. Using differential centrifugation of <i>Leishmania</i> subcellular structures, we showed that LinKAP was enriched in fractions colocalizing with other mitochondrial proteins. mNeonGreen labeling at the endogenous locus using CRISPR-Cas9 and confocal microscopy confirmed that LinKAP is a mitochondrial-associated protein in <i>Leishmania</i> but not specifically colocalized with kDNA. We cloned and expressed a truncated version of LinKAP (rLinKAP), containing part (15) of the several LinKAP amino acid repeats, demonstrating over 85% homology across <i>L. infantum, L. amazonensis, L. braziliensis,</i> and <i>L. mexicana</i> species. An adjuvanted formulation of LinKAP with Poly ICLC, a polyinosinic-polycytidylic acid (Poly I:C) stabilized with carboxymethylcellulose and polylysine, was used to vaccinate mice and hamsters as a prophylactic vaccine for visceral leishmaniasis. Animals immunized with rLinKAP showed a potent cellular and humoral response and a significant decrease in tissue parasitism when challenged with <i>L. infantum</i>. We also tested rLinKAP as a therapeutic vaccine in mice. Following therapeutic vaccination, antibody responses were enhanced, and cellular responses became apparent. Our treatment protocol inhibited splenic parasite burden by 75% in treated mice. In conclusion, our antigen discovery strategy and the observed protective effect highlight rLinKAP as a promising vaccine candidate for leishmaniasis.</p><p><strong>Importance: </strong>A previous reverse vaccinology study identified kinetoplast-associated protein-like protein from <i>Leishmania infantum</i> (LinKAP) as a potential new vaccine target, as this protein was recognized by the sera of protected mice in extracts of <i>Leishmania</i> promastigotes. Interestingly, LinKAP is a repetitive protein containing trichohyalin-plectin-homology (TPH) and TolA domains and was initially annotated as a kinetoplast-associated protein. We further characterized LinKAP as a mitochondrial-associated protein highly conserved among trypanosomatids. We also validated LinKAP as a promising vaccine antigen by using a truncated version of LinKAP (rLinKAP) as both a prophylactic and therapeutic vaccine, adjuvanted with Poly ICLC, to immunize animals against visceral leishmaniasis (VL). This disease, caused by the <i>Leishmania</i> parasite, affects several populations globally and still lacks highly effective vaccines. Identifying LinKAP and its preliminary characterization also provides new perspectives for studying its role in the parasite's biology.</p>","PeriodicalId":19052,"journal":{"name":"mSphere","volume":" ","pages":"e0009725"},"PeriodicalIF":3.7000,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12108087/pdf/","citationCount":"0","resultStr":"{\"title\":\"Characterization of a novel <i>Leishmania</i> antigen containing a repetitive domain and its potential use as a prophylactic and therapeutic vaccine.\",\"authors\":\"Bianca de Oliveira, Wanessa M Goes, Frederico C Nascimento, Juliana B T Carnielli, Eden R Ferreira, Alex Fiorini de Carvalho, Pablo Victor Mendes Dos Reis, Milton Pereira, Tiago Queiroga Nery Ricotta, Liliane Martins Dos Santos, Renan Pedra de Souza, Diego Esteban Cargnelutti, Jeremy C Mottram, Santuza R Teixeira, Ana Paula Fernandes, Ricardo T Gazzinelli\",\"doi\":\"10.1128/msphere.00097-25\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Human visceral leishmaniasis (HVL) is the second most lethal tropical parasitic disease. Currently, no prophylactic or therapeutic vaccines exist for HVL. Thus, the development of an efficacious vaccine is still needed. We previously performed an immunoproteomics analysis on <i>Leishmania amazonensis</i> parasite extracts to identify immunodominant antigens recognized by the sera of vaccinated and protected mice. Among the identified antigens, we discovered a novel, previously unstudied repetitive protein, initially annotated in <i>Leishmania</i> genomes as a kinetoplast-associated protein-like protein from <i>Leishmania infantum</i> (LinKAP), containing conserved domains (trichohyalin-plectin-homology [TPH] and TolA) that are associated with other mitochondrial proteins. LinKAP sequences are conserved across trypanosomatids, including <i>Endotrypanum, Leishmania,</i> and <i>Trypanosoma</i> species. Using differential centrifugation of <i>Leishmania</i> subcellular structures, we showed that LinKAP was enriched in fractions colocalizing with other mitochondrial proteins. mNeonGreen labeling at the endogenous locus using CRISPR-Cas9 and confocal microscopy confirmed that LinKAP is a mitochondrial-associated protein in <i>Leishmania</i> but not specifically colocalized with kDNA. We cloned and expressed a truncated version of LinKAP (rLinKAP), containing part (15) of the several LinKAP amino acid repeats, demonstrating over 85% homology across <i>L. infantum, L. amazonensis, L. braziliensis,</i> and <i>L. mexicana</i> species. An adjuvanted formulation of LinKAP with Poly ICLC, a polyinosinic-polycytidylic acid (Poly I:C) stabilized with carboxymethylcellulose and polylysine, was used to vaccinate mice and hamsters as a prophylactic vaccine for visceral leishmaniasis. Animals immunized with rLinKAP showed a potent cellular and humoral response and a significant decrease in tissue parasitism when challenged with <i>L. infantum</i>. We also tested rLinKAP as a therapeutic vaccine in mice. Following therapeutic vaccination, antibody responses were enhanced, and cellular responses became apparent. Our treatment protocol inhibited splenic parasite burden by 75% in treated mice. In conclusion, our antigen discovery strategy and the observed protective effect highlight rLinKAP as a promising vaccine candidate for leishmaniasis.</p><p><strong>Importance: </strong>A previous reverse vaccinology study identified kinetoplast-associated protein-like protein from <i>Leishmania infantum</i> (LinKAP) as a potential new vaccine target, as this protein was recognized by the sera of protected mice in extracts of <i>Leishmania</i> promastigotes. Interestingly, LinKAP is a repetitive protein containing trichohyalin-plectin-homology (TPH) and TolA domains and was initially annotated as a kinetoplast-associated protein. We further characterized LinKAP as a mitochondrial-associated protein highly conserved among trypanosomatids. We also validated LinKAP as a promising vaccine antigen by using a truncated version of LinKAP (rLinKAP) as both a prophylactic and therapeutic vaccine, adjuvanted with Poly ICLC, to immunize animals against visceral leishmaniasis (VL). This disease, caused by the <i>Leishmania</i> parasite, affects several populations globally and still lacks highly effective vaccines. Identifying LinKAP and its preliminary characterization also provides new perspectives for studying its role in the parasite's biology.</p>\",\"PeriodicalId\":19052,\"journal\":{\"name\":\"mSphere\",\"volume\":\" \",\"pages\":\"e0009725\"},\"PeriodicalIF\":3.7000,\"publicationDate\":\"2025-05-27\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12108087/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"mSphere\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1128/msphere.00097-25\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2025/4/22 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"mSphere","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1128/msphere.00097-25","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/4/22 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
Characterization of a novel Leishmania antigen containing a repetitive domain and its potential use as a prophylactic and therapeutic vaccine.
Human visceral leishmaniasis (HVL) is the second most lethal tropical parasitic disease. Currently, no prophylactic or therapeutic vaccines exist for HVL. Thus, the development of an efficacious vaccine is still needed. We previously performed an immunoproteomics analysis on Leishmania amazonensis parasite extracts to identify immunodominant antigens recognized by the sera of vaccinated and protected mice. Among the identified antigens, we discovered a novel, previously unstudied repetitive protein, initially annotated in Leishmania genomes as a kinetoplast-associated protein-like protein from Leishmania infantum (LinKAP), containing conserved domains (trichohyalin-plectin-homology [TPH] and TolA) that are associated with other mitochondrial proteins. LinKAP sequences are conserved across trypanosomatids, including Endotrypanum, Leishmania, and Trypanosoma species. Using differential centrifugation of Leishmania subcellular structures, we showed that LinKAP was enriched in fractions colocalizing with other mitochondrial proteins. mNeonGreen labeling at the endogenous locus using CRISPR-Cas9 and confocal microscopy confirmed that LinKAP is a mitochondrial-associated protein in Leishmania but not specifically colocalized with kDNA. We cloned and expressed a truncated version of LinKAP (rLinKAP), containing part (15) of the several LinKAP amino acid repeats, demonstrating over 85% homology across L. infantum, L. amazonensis, L. braziliensis, and L. mexicana species. An adjuvanted formulation of LinKAP with Poly ICLC, a polyinosinic-polycytidylic acid (Poly I:C) stabilized with carboxymethylcellulose and polylysine, was used to vaccinate mice and hamsters as a prophylactic vaccine for visceral leishmaniasis. Animals immunized with rLinKAP showed a potent cellular and humoral response and a significant decrease in tissue parasitism when challenged with L. infantum. We also tested rLinKAP as a therapeutic vaccine in mice. Following therapeutic vaccination, antibody responses were enhanced, and cellular responses became apparent. Our treatment protocol inhibited splenic parasite burden by 75% in treated mice. In conclusion, our antigen discovery strategy and the observed protective effect highlight rLinKAP as a promising vaccine candidate for leishmaniasis.
Importance: A previous reverse vaccinology study identified kinetoplast-associated protein-like protein from Leishmania infantum (LinKAP) as a potential new vaccine target, as this protein was recognized by the sera of protected mice in extracts of Leishmania promastigotes. Interestingly, LinKAP is a repetitive protein containing trichohyalin-plectin-homology (TPH) and TolA domains and was initially annotated as a kinetoplast-associated protein. We further characterized LinKAP as a mitochondrial-associated protein highly conserved among trypanosomatids. We also validated LinKAP as a promising vaccine antigen by using a truncated version of LinKAP (rLinKAP) as both a prophylactic and therapeutic vaccine, adjuvanted with Poly ICLC, to immunize animals against visceral leishmaniasis (VL). This disease, caused by the Leishmania parasite, affects several populations globally and still lacks highly effective vaccines. Identifying LinKAP and its preliminary characterization also provides new perspectives for studying its role in the parasite's biology.
期刊介绍:
mSphere™ is a multi-disciplinary open-access journal that will focus on rapid publication of fundamental contributions to our understanding of microbiology. Its scope will reflect the immense range of fields within the microbial sciences, creating new opportunities for researchers to share findings that are transforming our understanding of human health and disease, ecosystems, neuroscience, agriculture, energy production, climate change, evolution, biogeochemical cycling, and food and drug production. Submissions will be encouraged of all high-quality work that makes fundamental contributions to our understanding of microbiology. mSphere™ will provide streamlined decisions, while carrying on ASM''s tradition for rigorous peer review.